Mutational analysis of thymidylate synthase (TS) is proposed to probe structure-function relationships of this metabolically important enzyme. The synthase provides the cell with dTMP, a vital precursor specific to DNA synthesis. Because of its pivotal role in DNA replication TS is a target enzyme in chemotherapy. In order to understand these processes in greater depth it is of interest to study the regulation and mechanism of action of this enzyme. The overall objective of this study is to provide a genetic complement to the biochemical and structural studies being conducted on the synthase. As a basis to the proposed mutational analysis and having chosen the E. coli enzyme as a model system, we established the nucleotide sequence of the thyA gene, and the amino acid sequence of its TS product. Positive selection for thy- cells has allowed us to isolate a wide spectrum of random mutations after in vitro mutagenesis of the cloned gene. Rapid biochemical screening procedures as well as genetic and kinetic analyses of these mutants will more sharply define functional regions and will guide site-specific mutagenesis strategies to probe the precise involvement of particular residues in the catalytic process. The ability to construct strains which overproduce mutant enzyme will facilitate biochemical and structural studies in this and other laboratories. This combination of the genetic and biochemical approaches will help relate mutational change at the nucleotide and amino acid levels to altered kinetic and physical properties of TS. The ultimate goal is the delineation of those features and domains of TS which underly catalysis and are variously involved in substrate and cofactor binding, subunit folding, dimerization and multienzyme complex formation.